Intracellular Cl- activity (aiCl) was measured with double-barreled Cl- -selective microelectrodes in Triturus proximal tubule and the effects of ionic composition of the perfusion solutions and the cell membrane potentials were studied. In the absence of HCO-/3, aiCl averaged 22.4 +/- 0.4 mM, the value being 3.8 times that expected from the Nernst equation. Increases in peritubular HCO -/3 concentration without changing pH resulted in drastic decreases in aiCl. Addition of HCO-/3 to only the luminal fluid had no effect. Elimination of Na+ from the luminal fluid caused no immediate changes in aiCl, but long-lasting perfusions with a Na+-free solution resulted in a gradual decrease in aiCl. Lowering or raising intraluminal pH rapidly increased or decreased aiCl, respectively. Alanine-induced depolarization of the cell membranes exceeding 15-20 mV rapidly increased aiCl, and the increase was proportional to the driving force for Cl-. The results suggest the presence of a Cl-/OH- antiport mechanism in the luminal membrane and a Cl-/HCO-/3 exchange mechanism and voltage-dependent rectifying Cl- pathways in the peritubular membrane.